The usefulness of the determination of lipase activity in physiological fluids, including serum and duodenal fluid, for example, in early diagnosis of pancreatic diseases and monitoring the clinical course thereof has been generally acknowledge in the past. However, technical difficulties as well as problems with specificity and sensitivity of existing lipase methods have generally hindered the more widespread use of this enzyme as a primary laboratory indicator of pancreatic function.
The most widely accepted methods for measuring lipase activity in biological fluids usually employ olive oil as a substrate and rely on titration, with standardized sodium hydroxide of the fatty acids liberated during a 24 hour incubation period. Some major disadvantages of these types of methods include long incubation times, resulting in nonlinearity and reduced specificity; large sample requirements; the requirement for highly concentrated and stable olive oil emulsions, which are difficult and cumbersome to prepare; and the difficulties of reproducing the end point in the titration of the extremely weak long-chain fatty acids. Colorimetry of the free fatty acids produced by lipolysis of an olive oil substrate (in the form of their copper soaps, after extraction into a lipid solvent) permits shorter incubation times (10-30 minutes), but involves many tedious manipulations which limits its usefulness in a routine laboratory. The procedures described above, including variations thereof employing sensitive fluorescent pH indicators, for example, in addition to the described difficulties, lack the desired sensitivity and precision in the normal range of lipase activities.
In addition to the above described methods, turbidimetric methods of lipase analysis have been employed in the past, but the substrate concentrations used are suboptimal to permit the rate of substrate clearance to be measured in a reasonably accurate photometric range and initial increases in absorbance and nonlinear absorbance changes throughout the entire reaction period have been reported for patients' samples, indicating problems with some of these procedures. In addition to the olive oil substrate, several synthetic soluble chromogenic and fluorogenic substrates, mostly monoesters of long-chain fatty acids, have been proposed for use in the measurement of lipase activity in serum. However, it has been conclusively proved that pancreatic lipase does not act on soluble esters but is active only when adsorbed at an oil/water interface. Thus, the nonemulsified state of these types of substrates indicates limited usefulness as analytical tools for measuring pancreatic lipase activity. Lipase activity has also been determined by radial enzyme diffusion, based on measurement of the cross-sectional area of the clearing of an olive oil emulsion suspended in buffered agarose gel. The disadvantages of this method are that two hour incubation times are required, accurately measuring small diameters of the clear zone is difficult, and it is necessary to calibrate with secondary standards.
Thus, a method for the determination of lipase activity in biological fluids which has reduced incubation time, can employ relatively small amounts of sample fluids, and which is highly specific and sensitive is desirable. Further, a lipase activity measurement procedure which can be performed simply, using readily available laboratory equipment, would be advantageous.